Aryl sulfotransferase IV from rat liver has the broad substrate range that is characteristic of the enzymes of detoxication. With the standard assay substrates, 4-nitrophenol and 3'-phosphoadenosine 5'-phosphosulfate (PAPS), sulfation is optimum at pH 5.4 whereas the reaction is minimal in the physiological pH range. These properties preclude a physiological function for this cytosolic enzyme. Partial oxidation of the enzyme, however, results not only in an increase in the rate of sulfation but also in a shift of the pH optimum to the useful physiological pH range. The mechanism for this dependence on redox environment involves oxidation at Cys66, the cysteine residue that is conserved throughout the phenol sulfotransferase family. As documented by mass spectroscopic methods, oxidation of Cys66 by GSSG leads to the formation of an internal disulfide with Cys232; for mutants at Cys232, the product is a mixed disulfide of Cys66 and glutathione. Both disulfides are activated and enjoy an effective pH optimum. Although each of the five cysteines of the sulfotransferase subunit have been changed to serine, such mutant enzymes remain catalytically active. Only mutants at Cys66 are not activated and retain their acid pH optimum. The importance of these findings for an enzyme active in the detoxification of xenobiotics is that oxidation leads to changes in substrate specificity among phenols and to altered pH optima. It has now been shown that the sulfotransferases role in the detoxication process is subject to regulation by the normally present factors that apply under conditions of oxidative stress. As part of a program in which the enzymes that participate in the process of detoxication are being examined as to their catalytic mechanism, attention is being concentrated on the properties of two enzymes. One is a sulfotransferase (aryl sulfotransferase IV) which serves as a model for sulfotransferases in that it catalyzes the transfer of the sulfuryl group from 3-phosphoadenosine 5-phosphosulfate (PAPS) to a broad range of phenols; the products are 3-phosphoadenosine 5-phosphosulfate and the corresponding phenylsulfate. The enzyme has been cloned from rat liver and is expressed in very large quantities with E. coli. Expression, however, yields two different enzyme forms, only one of which, form B, is active in the physiological reaction, i.e. the formation of a phenylsulfate from PAPS. Labeled as forms A and B, both catalyze the reverse reaction, i.e. the formation of free phenol in the presence of PAP. It seems that PAP is bound to enzyme so tightly in form A, that the physiological donor, PAPS, is prevented from binding to the enzyme. Upon oxidation of protein thiols, PAP is released and the two forms of the enzyme can be interconverted. The effects oxidation and reduction of protein thiols appear to be physiological control mechanism for detoxication of phenols under conditions of oxidative stress.